Microfluidic mixer and microfluidic device comprising the same

ABSTRACT

The present invention relates to a microfluidic mixer and a microfluidic device including the same, and in the microfluidic mixer according to the present invention, a disk-shaped mixing unit with double U-shaped protruding portions formed therein can be continuously provided along a microchannel, thereby increasing collisions of samples to improve the binding efficiency thereof and shorten the binding time. Furthermore, the microfluidic device according to the present invention can detect a target material at high speed even at a high flow rate by including the microfluidic mixer, and thus can be usefully utilized for early diagnosis and prognosis diagnosis of a disease such as cancer.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to and the benefit of Korean PatentApplication No. 10-2020-0083881, filed on Jul. 8, 2020, the disclosureof which is incorporated herein by reference in its entirety.

BACKGROUND 1. Field of the Invention

The present invention relates to a microfluidic mixer and a microfluidicdevice comprising the same.

2. Discussion of Related Art

Exosomes are the smallest extracellular vesicles (30 to 150 nm), andplay a key role in intercellular signal transduction. Exosomes arepresent in human body fluids such as blood, saliva, and urine, and arevery important for clinical research because analysis of proteins andgenes in the exosomes can determine diagnostic and therapeutic methods.In general, patients with cancer have many exosomes derived from cancercells. By analyzing such cancer-specific exosomes, it is possible tograsp the degree of cancer progression and obtain information necessaryfor cancer treatment.

However, although the concentration of total exosomes in blood is ashigh as about 1×10^(10 to 11)/ml, cancer cell-derived exosomes that canbe used for actual cancer diagnosis are only about 1% of the totalexosomes. Therefore, there is a need for a technique capable ofselectively concentrating and detecting only target cancer cell-derivedexosomes from general exosomes having similar shapes and sizes.

As an exosome enrichment and detection method, an exosome isolation kit,ultracentrifugation, flow cytometry, and a nanotracking analysis, andthe like have been used to date, but these methods have limitations thatdevices are expensive, only skilled researchers can operate thesedevices, and it also takes a lot of time to operate these devices. Forthis reason, a microfluidic chip capable of isolating exosomes is beingactively developed worldwide.

SUMMARY OF THE INVENTION

An object of the present invention is to provide an ultra-high speedmicrofluidic mixer capable of shortening a binding time by efficientlybinding a sample even at a high flow rate.

Another object of the present invention is to provide a microfluidicdevice capable of continuously detecting a target material at high speedeven at a high flow rate based on excellent sample separation andconcentration performance.

Still another object of the present invention is to provide abioinformation analysis method for capturing a target material includedin the body using the microfluidic device.

To achieve the above objects, the present invention provides amicrofluidic mixer including: a first microchannel with a first inletand a second inlet, through which a fluid is introduced, being formed onone side and a first outlet, through which the fluid is discharged,being formed on the other side; and at least two or more disk-shapedmixing units disposed between the first inlet and second inlet and thefirst outlet, in which a U-shaped first protruding portion and secondprotruding portion protruding from the disk surface are formed in themixing unit and the first protruding portion is disposed inside a curvedportion of the second protruding portion.

Further, the present invention provides a microfluidic device including:the microfluidic mixer; a second microchannel with a first outlet of themicrofluidic mixer being connected to one side and a second outlet and athird outlet being formed on the other side; and at least two or moreseparating units disposed between the first outlet and the second inletand outlet.

In addition, the present invention provides a bioinformation analysismethod for capturing a target material included in the body.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and advantages of the presentinvention will become more apparent to those of ordinary skill in theart by describing in detail exemplary embodiments thereof with referenceto the accompanying drawings, in which:

FIG. 1 illustrates a perspective view of a microfluidic mixer accordingto an exemplary embodiment of the present invention;

FIG. 2 illustrates a top view of the microfluidic mixer according to anexemplary embodiment of the present invention;

FIG. 3 illustrates microchannel, separating unit, and branched channelportions in the configuration of a microfluidic device according to anexemplary embodiment of the present invention;

FIG. 4 exemplarily illustrates a method of using EpCAM and CD49fantibodies as markers to bind them to beads with different sizes, andthen separate and analyze them in order to predict the early diagnosisand prognosis of patients with cancer;

FIG. 5 exemplarily illustrates the process of separating andconcentrating exosomes, which are a target material, using themicrofluidic device according to the present invention as an exemplaryembodiment;

FIG. 6 is a set of images that capture fluorescence generated byinjecting fluorescent particles in order to evaluate the particle mixingefficiency of the microfluidic mixer according to the present invention;

FIG. 7 is a graph showing the mixing index (MI) values calculated byanalyzing fluorescent image pixels in order to confirm the degree towhich two types of particles are mixed;

FIG. 8 illustrates the results of measuring the binding efficiency ofantibody-coated beads and exosomes using the microfluidic mixeraccording to the present invention;

FIG. 9 illustrates the results of calculating the number of boundexosomes per bead in order to measure the binding efficiency ofantibody-coated beads and exosomes using the microfluidic mixeraccording to the present invention;

FIG. 10 illustrates the results of confirming the moving position ofeach particle in order to evaluate the separation efficiency by the sizeof the particles (microbeads) in a second microchannel 230 and aseparating unit 240 of a microfluidic device 10 according to the presentinvention using fluorescent particles;

FIG. 11 illustrates the results of evaluating the separation efficiencyof an actual sample according to the size of the particles (microbeads)in the second microchannel 230 and the separating unit 240 of themicrofluidic device 10 according to the present invention; and

FIGS. 12A and 12B show the results of verifying the separation andconcentration efficiency of the microfluidic device according to thepresent invention using exosomes extracted from cell lines and actualpatient plasma for separation and concentration of exosomes derived fromcancer.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Since the technology described below may be modified into various formsand include various exemplary embodiments, specific exemplaryembodiments will be illustrated in the drawings and described in detail.However, it is not intended to limit the technology described below tothe specific exemplary embodiments, and it is to be understood toinclude all the changes, equivalents and substitutions included in theidea and technical scope of the technology described below.

Terms such as first, second, A, and B may be used to describe variouscomponents, but the components should not be limited by the terms, andthe terms are used only for the purpose of distinguishing one componentfrom another. For example, without departing from the scope of thetechnology described below, a first component may be called a secondcomponent, and similarly, the second component may also be called thefirst component. The term and/or includes a combination of a pluralityof related listed items, or any item among the plurality of relatedlisted items.

Regarding the terms used herein, it is to be understood that singularexpressions include plural expressions unless the context clearlyindicates otherwise, and that the terms such as “comprise” are intendedto indicate that there is a feature, number, step, operation, component,part, or combination thereof described in the specification, but theterms do not exclude the possibility of the presence or the addition ofone or more other features or numbers, steps, operations, components,components, or a combination thereof.

Before giving a detailed description of the drawings, it is intended toclarify that the classification of the constituent parts in the presentspecification is merely division according to the main function eachconstituent part is responsible for. That is, two or more constituentparts described below may be combined into one constituent part, or oneconstituent part may also be divided into two or more and provided foreach more subdivided function. Moreover, of course, in addition to themain functions of each of the constituent parts described below areresponsible for, each constituent part may perform some or all of thefunctions that other constituent parts are responsible for and some ofthe main functions that each constituent part is responsible for may beexclusively performed by the other constituent parts.

In addition, in performing a method or operation method, each process ofthe above method may differ from the specified order unless a specificorder is explicitly stated in the context. That is, each process may beperformed in the same manner as the specified order, may be performed atsubstantially the same time, or may be performed in the reverse order.

Hereinafter, the present invention will be described in more detail withreference to the accompanying drawings to help understanding of thepresent invention. However, the following embodiments are provided foreasier understanding of the present invention, and the contents of thepresent invention are not limited by the following embodiments.

FIG. 1 illustrates a microfluidic device according to an exemplaryembodiment of the present invention. FIG. 2 illustrates a top view ofthe microfluidic mixer according to an exemplary embodiment of thepresent invention.

As illustrated in FIGS. 1 and 2, a microfluidic mixer 100 according tothe present invention includes: a first microchannel 140 with a firstinlet 110 and a second inlet 120, through which a fluid is introduced,being formed on one side and a first outlet 130, through which the fluidis discharged, being formed on the other side; and at least two or moredisk-shaped mixing units 150 disposed between the first inlet 110 andsecond inlet 120 and the first outlet 130, in which a U-shaped firstprotruding portion 160 and second protruding portion 170 protruding fromthe disk surface are formed in the mixing unit and the first protrudingportion 160 is disposed inside a curved portion of the second protrudingportion 170.

Different samples such as microbeads treated with a target material(exosomes, and the like) and a material that can bind to the targetmaterial (antibodies, and the like) may flow into the first inlet 110and the second inlet 120. The samples that have flowed into therespective inlets 110, 120 flow out to the first outlet 130 through themixing unit 150 along the first microchannel 140.

The mixing unit 150 may number two or more which may be disposed betweenthe first inlet 110 and second 120 and the outlet 130, and may be formedin the form of a disk such that a fluid including a sample maycontinuously and smoothly flow.

The mixing unit 150 has a two-layer structure in which a U-shaped firstprotruding portion 160 and second protruding portion 170 are doublyformed at the disk surface. A disk-shaped space is formed in the lowerportion of the mixing unit 150 which is directly connected to the firstmicrochannel 140, and thus generates a vortex of the fluid which flowsin, and the upper portion in which the U-shaped protruding space isformed (the first protruding portion and the second protruding portion)increases the number of collisions between the sample particles alongthe flow of the rising vortex. Even though the flow rate of the fluidflowing into the microfluidic mixer 100 is increased by the form of themixing unit 150, it is possible to prevent the particles of the samplefrom being aligned and remarkably shorten the binding time between thesamples.

As a specific exemplary embodiment, the mixing unit 150 may be disposedsuch that a direction in which both U-shaped ends of the firstprotruding portion and the second protruding portion face makes an angleof 10 to 170°, preferably 60 to 120°, and more preferably 80 to 100°with a direction in which the first microchannel extends, in order toincrease the number of collisions between sample particles generated inthe first protruding portion 160 and the second protruding portion 170.

As a specific exemplary embodiment, in the two or more mixing units 150,the first protruding portion 160 and the second protruding portion 170of adjacent mixing units 150 may be disposed such that both the U-shapedends face opposite directions. The first protruding portion 160 and thesecond protruding portion 170 are disposed such that both the U-shapeends face opposite directions, and thus serve to improve the mixingperformance of the microfluidic mixer 100 by inducing the circulatingflow of the vortex generated in the adjacent mixing unit 150. Asdescribed above, when the mixing units 150 are disposed, the fluid flowsinto the mixing unit 150, and then forms a vortex, and the samplescollide and bind with each other at the first protruding portion 160 andthe second protruding portion 170, and then a repetitive process may besmoothly and effectively performed by continuously connecting the flowof fluid moving to the next mixing unit 150.

As a specific exemplary embodiment, the length of the first microchannel140 between the two or more mixing units 150 may be shorter than thediameter of the disk-shaped mixing unit 150. When the length of thefirst microchannel 150 is longer than the diameter of the disk-shapedmixing unit 150, a pressure drop of the fluid may occur, andaccordingly, the flow in the mixing unit 150 is weakened, so that themixing efficiency of the sample may deteriorate.

FIG. 3 illustrates microchannel, separating unit, and branched channelportions in the configuration of a microfluidic device according to anexemplary embodiment of the present invention.

As illustrated in FIGS. 1 and 3, a microfluidic device 10 according tothe present invention includes: the microfluidic mixer 100; a secondmicrochannel 230 with the first outlet 130 of a microfluidic mixer 100being connected to one side and a second outlet 210 and a third outlet220 being formed on the other side; and at least two or more separatingunits 240 disposed between the first outlet 130 and the second outlet210 and third outlet 220.

As a specific exemplary embodiment, the separating unit 240 is forseparating the sample bound in the microfluidic mixer 100 according tosize, and includes a space that is wider than the width of the secondmicrochannel 230. Although there is no particular limitation, theseparating unit 240 is preferably rectangular.

Due to the continuous arrangement of the separating unit 240, sampleparticles with different sizes experience different inertial forces, andfinally, large sample particles move along the center of the secondmicrochannel 230 and small sample particles move along the side of thesecond microchannel 230 in different trajectories.

As a specific exemplary embodiment, the length of the secondmicrochannel 230 between the two or more mixing units 240 may be shorterthan the length of a width of the mixing unit 240. When the length ofthe second microchannel 230 between the separating units 240 is longerthan the length of a width of the separating unit 240, a pressure dropmay occur in the fluid passing through the second microchannel 230between the separating units 240, and accordingly, the flow in theseparating unit 240 is weakened, and thus, the separation efficiency ofthe sample may deteriorate.

As a specific exemplary embodiment, the second microchannel 230 may beone in which a first branched channel 250 which guides a portion of thesample separated via the separating unit 240 to the second outlet 210and a second branched channel 260 which guides the other portion of thesample to the third outlet 220 are formed.

In the first branched channel 250 and the second branched channel 260, abranch portion 200 may be branched downstream of a flow channel portion100 to guide the samples separated from the flow channel portion 100 todifferent outlets (the second outlet and the third outlet).

As a specific exemplary embodiment, the first branched channel 250 maybe provided in a straight line shape, and thus formed such that thecenter line coincides with the second microchannel 230, and the secondbranched channel 260 may be formed so as to be inclined in the outwarddirection of the second microchannel 230.

Specifically, the first branched channel 250 may guide a portion of thesamples concentrated at the center in the second microchannel 230, forexample, a sample having a relatively large size to the second outlet210, and may guide the remaining samples of the samples moving to atleast one side in the second microchannel 230, for example, a samplehaving a relatively small size, to the third outlet 220.

For this, the first branched channel 250 may be provided in a straightline shape, and may be formed such that the center line of the firstbranched channel 250 coincides with the center line of the secondmicrochannel 230, so that the second microchannel 230 and the secondoutlet 210 provided at the end of the first branched channel 250 may bedisposed to be spaced apart from each other on the same line.

In contrast, the second branched channel 260 may include a portionformed so as to be inclined from the downstream of the secondmicrochannel 230 to the outside of the second microchannel 230, aportion formed straight, and a portion formed so as to be inclinedtoward the third outlet 220 disposed on the same line as the secondoutlet 210, along the flow direction of a non-Newtonian fluid or sample.

In particular, as illustrated in FIG. 3, when two second branchedchannels 260 are provided, the first branched channel 250 may bedisposed between the two second branched channels 260. Moreover, the twosecond branched channels 260 are formed symmetrically with each otherwith the first branched channel 250 therebetween, and thus, may becombined at the third outlet 220.

Further, the width of the first branched channel 250 may be formed to benarrower than the width of the second microchannel 230, whereas thewidth of the second branched channel 260 may be formed to be wider thanthe width of the second microchannel 230. Moreover, the length of thefirst branched channel 250 may be formed to be shorter than the lengthof the second microchannel 230, and the length of the second branchedchannel 260 may be formed to be longer than the length of the firstbranched channel 250.

A portion of the samples concentrated at the center in the secondmicrochannel 230, for example, a sample having a relatively large sizemay be relatively rapidly collected or concentrated in the second outlet210, and the remaining samples of the samples moving to at least oneside in the second microchannel 230, for example, a sample having arelatively small size may be relatively slowly collected in the thirdoutlet 220, and then discharged outside.

Additionally, by providing a suction flow to the second outlet 210, itis possible to improve the concentration proportion of the samplecollected in the first outlet 210, for example, a sample having arelatively large size,

Meanwhile, the microfluidic device according to the present inventionmay be utilized for a bioinformation analysis method for capturing atarget material included in the body

Epithelial to mesenchymal transition (EMT) means a transition fromepithelial cell properties to mesenchymal cell properties, andEMT-related reprogramming of cells is closely related not only tochanges in various regulatory networks, but also to close interactionsbetween these networks.

Since CD49f and EpCAM on the cell surface have EMT characteristics usingstem cell markers. EpCAM and CD49f antibodies are used as markers tobind these antibodies to beads with different sizes, and then the earlydiagnosis and prognosis of patients with cancer may be predicted byseparating and analyzing the antibodies, as illustrated in FIG. 4.

As illustrated in FIG. 5, when 7 μm beads bound to the EpCAM antibodyand 15 μm beads bound to the CD49f antibody are injected into the firstinlet 100 and an exosome sample is injected into the second inlet 120 bya syringe pump in the microfluidic device according to the presentinvention as an exemplary embodiment, the injected exosomes will bind tothe antibody on the bead surface while passing through the microfluidicmixer 100. Bound exosomes move to the separating unit 240 via the secondmicrochannel 230, 15 μm beads, which are relatively large, move alongthe center of the second microchannel 230, and thus are separated intothe second outlet 210, and 7 μm, which are relatively small, areseparated into both sides, and thus are separated into the third outlet220, so that exosomes expressing different surface proteins can beseparated and concentrated.

FIG. 6 is a set of images that capture fluorescence generated byinjecting fluorescent particles in order to evaluate the particle mixingefficiency of the microfluidic mixer according to the present invention.

FIG. 7 is a graph showing the mixing index (MI) values calculated byanalyzing fluorescent image pixels in order to confirm the degree towhich two types of particles are mixed.

100 nm green fluorescent particles and 7 μm blue and 15 μm redfluorescent particles were injected into the microfluidic mixeraccording to the present invention, and the mixing efficiency wasmeasured at a flow rate of 50 to 200 μl/min.

The MI range is between 0 and 1, and has a value of 1 when the particlesare perfectly mixed. It can be confirmed that the MI value of 100 nm and7 μm particles increases as the cycle increases from 1 to 15 over theentire flow rate range. It can be seen that the MI value of 15 μmparticles decreases at a flow rate of 200 μl/min. In a microfluidicchannel environment, as the particle size increases, the particleexperiences more inertial force, and as the flow rate increases, theforce experienced by the particle increases with the square root. Theflow rate range that may be used in the microfluidic mixer of thepresent invention is 50 to 150 μl/min.

FIG. 8 illustrates the results of measuring the binding efficiency ofantibody-coated beads and exosomes using the microfluidic mixeraccording to the present invention. FIG. 9 illustrates the results ofcalculating the number of bound exosomes per bead in order to measurethe binding efficiency of antibody-coated beads and exosomes using themicrofluidic mixer according to the present invention. In order toconfirm the binding efficiency of exosomes and beads in the microfluidicmixer according to the present invention, an antibody-coated beadbinding experiment was performed using exosomes derived from a cellline. The experiment was performed by extracting exosomes from SK-BR-3,which is a cell line with high EpCAM expression, and MDA-MB-231, whichis a cell line with high CD49f expression. The initial concentrationsand sizes of exosomes with high EpCAM and CD49f expression were measuredusing a nanoparticle tracking analyzer (NTA). The maximum number ofexosomes which can be captured per microbead is defined as N_(exo), andthe calculation formula is as follows [Equation 1].

N _(exo) =SA _(bead) /CS _(exo)  [Equation 1]

In [Equation 1], an exosome surface cross-sectional area (CS_(exo)) is2rπ2 (NTA measurement average diameter: 200 nm), and a bead surface area(SA_(bead)) is 4πr2 (bead diameter: 7 μm, 15 μm). Based on the number ofexosomes 10⁹, the number of beads was set in a range of N_(exo)=10⁹,10⁸, and 10⁷. EpCAM antibody-coated 7 μm beads and EpCAM-expressingexosomes were allowed to pass through the microfluidic mixer accordingto the present invention at a flow rate of 150 μl/min, and the exosomesbound to the beads were calculated by measuring the exosomes remainingafter the experiment by NTA. Experiments were also performed onCD49f-coated 15 μm beads and CD49f-expressing exosomes in the samemanner. As illustrated in FIG. 9, the EpCAM-expressing exosomes had thehighest binding efficiency of 89.99% when N_(exo) was 10⁹. TheCD49f-expressing exosomes also had the highest binding efficiency of94.63% when N_(exo)=10⁹.

FIG. 10 illustrates the results of confirming the moving position ofeach particle through a fluorescence experiment in order to evaluate theseparation efficiency by the size of the particles (microbeads) in asecond microchannel 230 and a separating unit 240 of a microfluidicdevice 10 according to the present invention using fluorescentparticles. The fluorescence experiment was performed by making a changein the dimensionless Reynolds number (Re) value, which is a ratio ofinertial force to viscous force. The formula is as follows [Equation 2].

Re=ρVd/μ  [Equation 2]

In [Equation 2], ρ is the density of a fluid, μ is the viscosity of thefluid, V is a maximum flow rate, and d is a hydraulic length. Theexperiment was performed while varying flow rate and viscosity. Asillustrated in FIG. 10, it can be confirmed that the 15 μm beads withlarge particles are arranged in the center at all flow rates, and theposition of the 7 μm bead changes from the outside to the inside as theRe value increases.

FIG. 11 illustrates the results of evaluating the separation efficiencyof an actual sample according to the size of the particles (microbeads)in the second microchannel 230 and the separating unit 240 of themicrofluidic device 10 according to the present invention.

In order to confirm the bead separation efficiency in an actual sample,beads to which an antibody used in the actual sample was bound wereused, and an experiment was performed using phosphate buffered saline(PBS), which is similar to the components of human body fluids, and anactual plasma solution. As illustrated in FIG. 11, it was confirmed thatboth the PBS solution and the serum had the highest separationefficiency at a flow rate of 150 μl/min.

FIG. 12 shows the results of verifying the separation and concentrationefficiency of the microfluidic device according to the present inventionusing exosomes extracted from cell lines and actual patient plasma forisolation and concentration of exosomes derived from cancer.

In order to confirm the separation efficiency of EpCAM and CD49f, beadswith different sizes, whose surface was coated with an antibody capableof capturing the exosomes were injected into a microfluidic device 10.Culture solutions of MCF-7 and SK-BR-3, which are cell lines with highEpCAM expression, and MDA-MB-231 and Hs578T, which are cell lines withhigh CD49f expression, were used, and EpCAM-positive (EpCAM+) exosomesand CD49f-positive (CD49f+) exosomes were used. At the same time, inorder to compare the separation and concentration efficiencies of themicrofluidic device 10 according to the present invention, exosomes werealso separated by a precipitation method using polyethylene glycol amongconventionally used exosome separation methods. For the separatedexosomes, the relative expression levels of EpCAM and CD49f compared toCD63 mRNA, which represents the entire exosomes, were verified usingreal-time PCR using a target specific pre-amplification method.

As illustrated in FIG. 12, it was revealed that the EpCAM mRNA level inthe exosomes separated by the microfluidic device 10 according to thepresent invention was 15.7-fold higher on average than that in theexosomes separated by the precipitation method. Likewise, the CD49f mRNAlevel in exosomes separated by the microfluidic device 10 was found tobe 40-fold higher on average than that in exosomes separated by theprecipitation method (FIG. 12A). The separation efficiencies when amethod using the microfluidic device 10 and the precipitation methodwere used were evaluated using 100 μl of plasma of each of a healthycontrol and patients with breast cancer. In the case of patients withbreast cancer, the microfluidic device 10 was found to havesignificantly higher expression of EpCAM and CD49f mRNA than aprecipitation-based separation method (FIG. 12B). Since EpCAM is knownas a tumor-specific marker and the expression thereof is low undernormal conditions, the level of EpCAM in the healthy control was foundto be low in both methods as expected.

From this, it can be seen that the method using the microfluidic device10 according to the present invention can separate and concentrate EpCAMand CD49f-expressing exosomes more efficiently than the precipitationmethod.

In the microfluidic mixer according to the present invention, adisk-shaped mixing unit with double U-shaped protruding portions formedtherein can be continuously provided along a microchannel, therebyincreasing collisions of samples to improve the binding efficiencythereof and shorten the binding time. Furthermore, the microfluidicdevice according to the present invention can detect a target materialat high speed even at a high flow rate by including the microfluidicmixer, and thus can be usefully utilized for early diagnosis andprognosis diagnosis of a disease such as cancer.

The above-described description of the present invention is provided forillustrative purposes, and those skilled in the art to which the presentinvention pertains will understand that the present invention can beeasily modified into other specific forms without changing the technicalspirit or essential features of the present invention. Therefore, itshould be understood that the above-described embodiments are onlyexemplary in all aspects and are not restrictive.

What is claimed is:
 1. A microfluidic mixer comprising: a firstmicrochannel with a first inlet and a second inlet, through which afluid is introduced, being formed on one side and a first outlet,through which the fluid is discharged, being formed on the other side;and at least two or more disk-shaped mixing units disposed between thefirst inlet and second inlet and the first outlet, wherein a U-shapedfirst protruding portion and second protruding portion protruding fromthe disk surface are formed in the mixing unit and the first protrudingportion is disposed inside a curved portion of the second protrudingportion.
 2. The microfluidic mixer of claim 1, wherein the mixing unitis disposed such that a direction in which both the U-shaped ends of thefirst protruding portion and the second protruding portion face makes anangle of 10 to 170° with a direction in which the first microchannelextends.
 3. The microfluidic mixer of claim 1, wherein in the two ormore mixing units, the first protruding portion and the secondprotruding portion of adjacent mixing units are disposed such that boththe U-shaped ends face opposite directions.
 4. The microfluidic mixer ofclaim 1, wherein a length of the first microchannel between the two ormore disk-shaped mixing units is shorter than a diameter of thedisk-shaped mixing unit.
 5. A microfluidic device comprising: themicrofluidic mixer of any one of claim 1; a second microchannel with thefirst outlet of the microfluidic mixer being connected to one side and asecond outlet and a third outlet being formed on the other side; and atleast two or more separating units disposed between the first outlet andthe second outlet and third outlet.
 6. The microfluidic device of claim5, wherein separating unit is rectangular.
 7. The microfluidic device ofclaim 5, wherein a length of the second microchannel between the two ormore separating units is shorter than a length of a width of theseparating unit.
 8. The microfluidic device of claim 5, wherein in thesecond microchannel, a first branched channel which guides a portion ofa sample separated via a separating unit to the second outlet and asecond branched channel which guides the other portion of the sample tothe third outlet are formed.
 9. The microfluidic device of claim 8,wherein the first branched channel is provided in a straight line shape,and thus formed such that a center line coincides with the secondmicrochannel, and the second branched channel is formed so as to beinclined in the outward direction of the second microchannel.
 10. Abioinformation analysis method for capturing a target material comprisedin the body using the microfluidic device of claim 5.